Field of the Invention
The invention relates to a functional human brown adipocytes population, in which the expression of UCP1, CIDEA, CPT1B and Bcl-2 is higher, the expression of Bax is lower, and the expression of PPARα, PGC-1α, PGC-1β and PRDM16 is similar compared with the corresponding expressions of a human white adipocytes population. The invention also relates to a method for differentiating hMADS cells into the functional human brown adipocytes population, a method for converting a human white adipocytes population into the functional human brown adipocytes population, and a screening method for molecules capable of modulating the body weight in an individual.
Description of Related Art including information disclosed under 37 CFR 1.97 and 1.98
White adipose tissue (WAT) plays a key role in humans in the control of energy balance and carbohydrate-lipid homeostasis (Ailhaud G. et al., Annu Rev Nutr, 1992. 12: p. 207-233; Rosen, E. D. and B. M. Spiegelman, Nature, 2006. 444(7121): p. 847-53). As opposed to WAT, brown adipose tissue (BAT) is specialized in adaptive thermogenesis during which the uncoupling protein UCP1 plays a decisive role. The presence of brown adipose tissue is well known in rodents as in larger newborn mammals (Garruti, G. and D. Ricquier, Int J Obes Relat Metab Disord, 1992. 16(5): p. 383-90; Cannon, B. and J. Nedergaard, Physiol Rev, 2004. 84(1): p. 277-359). A particularly striking fact is that recent data suggest the existence of functional BAT in healthy adult humans (Nedergaard, J. et al., Am J Physiol Endocrinol Metab, 2007. 293(2): p. E444-52; Cypess, A M et al., N. Engl. J. Med. 2009. 360: p. 1509-17; Saito, M. et al., Diabetes 2009. Published Ahead of Print, Online April 28; van Marken Lichtenbelt, W et al., N. Engl. J. Med. 2009. 390: p. 1500-08; Virtanen, K A et al., N. Engl. J. Med. 2009. 360: p. 1518-1525).
In vivo, the appearance in rodents of brown adipocytes islets in the middle of WAT deposits is known (Timmons, J. A., et al., Proc Natl Acad Sci USA, 2007. 104(11): p. 4401-6; Cousin, B., et al., J Cell Sci, 1992. 103 (Pt 4): p. 931-42; Xue, B., et al., Mol Cell Biol, 2005. 25(18): p. 8311-22), following an exposure to cold or a treatment with β-adrenergic receptor (β-AR) agonists. Similarly, after a treatment with PPARγ agonistic ligands, the appearance of cells expressing UCP1 in white adipose deposits was also documented in rodents and in human (Wilson-Fritch, L., et al., J Clin Invest, 2004. 114(9): p. 1281-9; Fukui, Y., et al., Diabetes, 2000. 49(5): p. 759-67; Bogacka, I., et al., Diabetes, 2005. 54(5): p. 1392-9). However, these observations cannot exclude the prior existence of brown precursors in such sites. Reciprocally, the rapid conversion of BAT into WAT in human newborns as in ovine and bovine newborns cannot exclude the prior existence of white precursors (Casteilla, L., et al., Am J Physiol, 1987. 252(5 Pt 1): p. E627-36; Casteilla, L., et al., Biochem J, 1989. 257(3): p. 665-71; Casteilla, L., et al., Eur J Biochem, 1991. 198(1): p. 195-9). The existence of a precursor cell common to white and brown lines remains to be shown, insofar as the preadipocytes obtained ex vivo from WAT and BAT give only rise to white and brown adipocytes, respectively (Moulin, K., et al., Biochem J, 2001. 356(Pt 2): p. 659-64). Recent work in the mouse supports this possibility by showing the existence of a myogenic transcriptome signature of brown adipocytes distinct from that of white adipocytes (Timmons, J. A., et al., Proc Natl Acad Sci USA, 2007. 104(11): p. 4401-6).
However, other work underlines the possibility to generate brown adipocytes from white adipocytes by transgenesis (Tiraby, C. and D. Langin, Trends Endocrinol Metab, 2003. 14(10): p. 439-41; Tiraby, C, et al., J Biol Chem, 2003. 278(35): p. 33370-6) and several co-activators and transcription factors take part in the formation of brown adipocytes. Thus, during differentiation PGC-1α and PGC-1β play an essential and complementary role in mitochondriogenesis and respiration (Puigserver, P., et al., Mol Cell, 2001. 8(5): p. 971-82; Uldry, M., et al., Cell Metab, 2006. 3(5): p. 333-41). However, contrary to these PPARγ coactivators, the zinc finger transcription protein PRDM16 truly controls the “brown” determination of white preadipocytes by induction of PGC-1α, UCP1 and type II iodothyronine deiodinase (Dio2) (Seale, P., et al., Cell Metab, 2007. 6(1): p. 38-54).
The article by Zilberfarb et al., 1997, J Cell Sci 110(Pt 7), 801-807, describes an immortalized line of human brown adipocytes, PAZ6, which is obtained by transgenesis. This article shows that the PAZ6 line expresses mRNA coding for UCP1.
However, it does not show that this expression leads to a functional UCP1, i.e., one that has uncoupling activity due to its location in the inner mitochondrial membrane, thus conferring to the human brown adipocytes a respiratory activity and an uncoupling activity that are significantly higher than those of human white adipocytes. Moreover, no stimulation of respiratory activity by a specific agonist of β-adrenergic receptors is reported as being dependant on the presence of UCP1.
Moreover, the article by Zilberfarb et al., 1997 describes that the β3-adrenergic receptor is coupled with adenylate cyclase and with lipolysis in PAZ6 cells. This coupling between β3-adrenergic receptor and adenylate cyclase is known to be preliminary to a cascade of events leading to the uncoupling activity of UCP1.
However, the article by Zilberfarb et al., 1997 also describes that similar results of coupling between adenylate cyclase and β3-adrenergic receptor have been obtained by other authors (Murphy at al., 1993) by using freshly isolated rat adipocytes. These are in fact white adipocytes (see Murphy et al., 1993: “Correlation of β3-adrenoreceptor-induced activation of cyclic AMP dependent protein kinase with activation of lipolysis in rat white adipocytes”).
Thus, the results obtained and described in Zilberfarb et al., 1997 with brown PAZ6 adipocytes are similar to those obtained with white adipocytes, which inevitably leads to a respiratory activity and an uncoupling activity from these brown adipocytes similar to those of white adipocytes. The brown PAZ6 adipocytes described in Zilberfarb et al., 1997 are thus not functional in the context of the present invention.
Recently we have isolated mesenchymatous stem sells (human multipotent adipose-derived stem cells, or hMADS cells) from human adipose tissue. In the clonal state, these cells have a normal karyotype, a strong self-renewal capacity, and an absence of tumorigenicity. hMADS cells prove to be capable of differentiating into several lineages, in particular osteoblastic and adipocytic, and lead in vivo to a bone and muscle regeneration (Rodriguez, A. M., et al., Biochem Biophys Res Commun, 2004. 315(2): p. 255-63; Rodriguez, A. M., et al., J Exp Med, 2005. 201 (9): p. 1397-405; Zaragosi, L. E., et al., Stem Cells, 2006. 24(11): p. 2412-9; Elabd, C., et al., Biochem Biophys Res Commun, 2007. 361(2): p. 342-8). Once differentiated into adipocytes, hMADS cells acquire the functional properties of human white adipocytes (secretion of leptin and adiponectin, responses to insulin and to β-adrenergic agonists and, specific to primates, to atrial natriuretic factor, Rodriguez, A. M., et al., Biochem Biophys Res Commun, 2004. 315(2): p. 255-63). hMADS cells thus represent a suitable cellular model to study their possible capacity to also differentiate into brown adipocytes.
Our results show that a prolonged chronic activation of PPARγ is sufficient to promote their conversion in vitro and to increase their respiratory and uncoupling capacities, and that β-adrenergic receptor agonists, including β3 agonists, positively modulate the expression of UCP1 as well as the stimulation of respiratory activity by a specific agonist of β-adrenergic receptors such as isoproterenol. The differentiation of hMADS cells leads to a functional human brown adipocytes population which can be used as a cellular model, notably to identify molecules capable of modulating body weight, and in particular to treat excess weight and/or obesity.